MARK/PAR1 kinases occupy critical signaling nodes in the development of higher organisms, and have been linked to a plethora of disease states including Autism, Alzheimer's, cancer, and heart disease. The goal of this proposal is to elucidate the structural and functional role of the kinase-associated 1 (KA1) domain, which resides at the C-terminus of MARK/PAR1 and related kinases. This module has recently been shown to interact with both phospholipid and protein ligands, but has also been proposed to provide an autoinhibitory function for the attached kinase domain. Preliminary studies have established that a separately purified KA1 module does bind the MARK1 kinase domain and can inhibit activity in vitro. We hypothesize that kinase autoinhibition is relieved upon binding o the KA1 domain to lipid and/or protein ligands and plan to rigorously assess these features of MARK/PAR1 kinases. The aims of this project are to (i) fully characterize the interaction between the MARK1 kinase and KA1 domains through a battery of structural and biophysical methods and, and (ii) establish a functional role for the KA1 domain in MARK1 activity by in vitro kinetic and in vivo cell-based methods, including determination of whether exposure to lipid or protein ligands of this domain disrupt autoinhibition and activate the kinase. These studies will shed light onto how varied inputs from lipid or protein ligands can lead to kinase regulation, and will provide the groundwork for design of specific allosteric inhibitors of MARK/PAR1 kinases.